臺灣博碩士論文加值系統

世界各國飼料蛋白嚴重不足，每年需進口大量的魚粉以滿足需要。隨著世界魚類資源的減少，國際魚粉市場供應也日趨緊張，不可能長期依靠進口魚粉來彌補飼料蛋白的不足。 隨著家禽業的蓬勃發展，飼料原料(尤其是蛋白飼料)的缺乏已成為非常突出的問題。綠藻是潛在新產品和新用途的生物資源中最重要ㄧ環，綠藻中存在著豐富的結構獨特的有機化合物，除了代謝產物具有多種多樣的生物活性，培養後產生的藻體與衍生物，將可作為動物飼料或生質能源等用途，使綠藻能成為非傳統蛋白質的來源。因此，加強綠藻單細胞蛋白的研究與開發，利用現代微生物技術開發無糧型高效能蛋白營養飼料，其市場前景將十分廣闊。
由於大多的綠藻細胞壁堅硬，需經過物理機械法或有機溶劑萃取等耗能又不環保之步驟，破壁後的綠藻亦無法有效提供禽畜動物的全營養價值。為了提高綠藻飼糧利用率，酶溶法(纖維素酶、半纖維素酶與脂酶等)因其作用條件最為溫和，細胞壁損壞的程度可以控制，內含物成分不易受到破壞，目前是最適合應用在多種單細胞生物的細胞降解(如綠藻的破壁)。而富含纖維素分解酵素（cellulase）的微生物中，又以真菌類為最多。根據文獻回顧指出，早在近10年內，就有國內外學者相繼將秀珍菇菌其中分泌的酵素應用在特定物質(如木質素)，並將農業廢棄物轉換為動物飼料，有鑑於上述提及的綠藻產業上應用的瓶頸乃出於其堅硬的細胞壁保護使藻內營養素無法有效釋出，而秀珍菇菌本身也具有分泌纖維水解酵素的能力，故本論文研究目的欲評估是否可將秀珍菇菌應用在水解綠藻細胞壁上。同時，在綠藻種的選取上，考量其新穎性與適合度，選取常用於水產養殖相關產業的扁藻。而本論文主軸乃利用微生物破壁(降解法)促進綠藻細胞壁水解與提高可溶性內含物析出。
本研究之結果顯示，扁藻的添加比例與秀珍菇菌的生長速度成正比，使用擴培七天的秀珍菇菌菌液對於菌的生長與藻的水解較為適當。擴培過的秀珍菇菌確實可分泌內切型的纖維水解酵素來水解扁藻當作營養來源促進生長。而在扁藻內含物析出試驗中，MYC培養基(葡萄糖存在下)中的秀珍菇菌液能顯著提高培養液中的葉綠素、粗脂肪、可溶性蛋白質及游離胺基酸含量，特別是一些胺基酸如麸胺酸(Glutamic acid)、丙胺酸(Alanine)及離胺酸(Lysine)等。在掃描式電子顯微鏡(SEM)的檢查下，扁藻在秀珍菇的作用時間(10天)下，可看到細胞壁有嚴重破裂的情況，且較傳統使用的超音波震(sonication)破壁法的效果為佳。從本研究結果得知，若利用特定真菌與綠藻共同培養方式，可提高微生物降解(水解)綠藻細胞壁的反應速率，相信可更易為人體或畜禽所高度吸收及完全利用，對綠藻保健食品的開發及飼料營養添加劑有相當大的開發前景。

For the biological resource development of algae, to explore algae to serve as renewable energy sources (such as biodiesel and bio-ethanol) is feasible. Especially the rapid growth of microalgae, shows efficient utilization on solar energy more than other grains. In particular, in recent years, global demand for energy, and vigorously developing the traditional agricultural crops (soybeans and corn, etc.) as raw materials for biomass energy sources, coupled with the outbreak of diseases such as avian influenza and foot and mouth disease, resulted in livestock demand for feed growing. In addition to fatty acids and lipids, microalgae also contain a large number of non-nitrogen-containing organic compounds (starch, mannitol, etc.) and several proteins (up to 60%), which were identified as having potential to replace soybean (containing 45% protein) as a non-traditional protein animal alternative feed ingrecient and nutrient additives.
Because the thickened cell wall of most microalgae, livestock can not directly absorbed lots of nutrients from microalgae. In order to elevate the microalgae value-adding, enzyme dissolution method (cellulase, hemicellulase and lipase, etc.) is currently the most suitable for application in a variety of microorganisms (such as broken green algae cell wall) as the mechanical method, or organic solvent extraction caused highly energy consumption and environmental pollution, which also can not effectively provide the full nutrient value to animal. The research project will mainly focus on specific microbiological methods for microalgae degradation, which is currently the most suitable for applications in a variety of single-celled organisms, (such as green algae). Fungi are microorganisms which contain rich cellulose enzymes (cellulase). Therefore, this study intends to use the edible fungi/algae co-culture system (such as Pleurotus ostreatus and Pleurotus eryngii) to explore the more efficient degradation of Chlorella (Chlorellaceae spp. and Tetraselmis spp.) cell wall method by a variety of cellulase. Under a variety of bacteria/algae ratio, the morphology of Chlorella was observed by microscopy examination, and the isolated extracts from green algae, including chlorophyll, proteins, peptides, free amino acids and crude lipids were measured at short-term (0-72 hr) and long-term (14 day) exposure.
With the release analysis of the composition of Tetraselmis spp., we found that the chlorophyll, fat, soluble protein and free amino acids, in particular, some essential amino acids such as glutamic acid, alanine and lysine were significantly increased in the MYC medium (the presence of glucose) containing Pleurotus ostreatus. By the scanning electron microscopy (SEM) examination, the broken cell wall of Tetraselmis spp. following 10 days exposure of Pleurotus ostreatus was observed, more than with ultrasound breakdown method. Experimental results demonstrated that the microbial degradation (hydrolysis) method could facilitate the reaction rate of the Chlorella cell wall breakdown, and provide a novel and available health food and feed nutrient additives for the use of human being or animal.

中文摘要………………………………………………………………………………I
英文摘要………………………………………..……………………………………III
致謝………………………………………..………………………………………….V
目次………………………………………………………………………………… VI
圖目錄……………………..………………………………………………………VIII
表目錄……………………………………………………………………………… IX
縮寫表.……………………….……………………….…………………………….. X
一、文獻回顧……………………….……………………….…………………..……1
1. 藻類應用概述……………………….……………………….………….…………1
1.1 水產養殖上的應用…………………………………………….…………………1
1.2 醫藥食品上的應用…………………………………………….…………………2
1.3 生質能源的應用……………………….…………………….…...………………2
2. 綠藻的生長與分部……………………….……………………….……………….3
2.1 本論文所使用之藻類簡介………………….…………………….……………...4
3. 綠藻的營養素……………………….…………………….…………………….…5
3.1 綠藻對人體的影響及其保健功效………………….…………………….……...6
3.2 禽畜動物飼料的需求………………….…………………….…………………...8
4. 綠藻的細胞壁結構與綠藻工業待解決之問題………………….………………..8
4.1 現行一般綠藻工業上所採行的破壁方式………………….……………………9
5. 酵素水解綠藻細胞壁之研究………………….……………...……………….…10
5.1 纖維素分解酵素應用於藻類之研究………………….…………………….….11
6. 具纖維稅解酵素分泌能力之食用真菌………………….………………………12
6.1 真菌之簡介………………….…………………….………………………….…12
6.2秀珍菇之簡介………………….…………………….………………………..…13
二、研究方法………….…………………….……………………….………………15
1. 實驗材料…………………………….…………….…………….………………..16
1.1 真菌株………….…………………….……………………….………………....16
1.2 設備………….…………………….……………………….……………………16
1.3實驗方法……………………….……...…………………….…………………...17
1.3.1真菌菌絲體的培養………….………….……….……………………….…….17
1.3.2藻類的培養…………………………….………………………………………19
1.3.3微生物(真菌)分解綠藻細胞壁之相關試驗….………….……………………21
1.3.4 數據統計與分析( Data Analysis )...……...………………………………….. 26
三、結果…………………………………………………………….………….……28
1. 扁藻對秀珍菇菌在平盤試驗中生長之影響…………………………………….27
2. 扁藻與秀珍菇菌在搖瓶液態培養中生長之關係……………………………….27
3. 培養基中葡萄糖含量對秀珍菇菌水解扁藻細胞壁之影響…………………….28
4. 比較外力式與酵素式破壁法對水解扁藻細胞壁之差異……………………….28
四、討論………………...…………………………………………………………….29
五、結論………………...…………………………………………………………….34
六、結果圖表…………………………………………………………………………35
七、參考文獻…………………………………………………………………………51

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